Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Pyruvate Kinase”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 55 records · Page 3Linked to original sources
PubMed1991

The role of protein kinases in anoxia tolerance in facultative anaerobes: purification and characterization of a protein kinase that phosphorylates pyruvate kinase.

A protein kinase which phosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia-tolerant gastropod mollusc Busycon canaliculatum. Purification involved four steps: poly(ethylene glycol) fractionation, affinity chromatography on Blue agarose, ion-exchange chromatography on phosphocellulose and preparative isoelectric focusing (pI = 5.5). The activity was monitored by following changes in pyruvate kinase I50 values for L-alanine which have previously been linked to changes in the degree of enzyme phosphorylation. The correlation between enzyme phosphorylation and changes in the L-alanine inhibition constant was also directly demonstrated in the present paper by radioactively labelling PK with [tau-32P]ATP. The final purified protein kinase solution gave a single band on SDS-gel electrophoresis with a molecular weight of 37,000 +/- 2000. Kinetic analysis of the purified protein kinase (PK-kinase) showed a pH optimum of 7.0, an absolute requirement for magnesium ions (Km = 1.29 mM), a relatively high affinity for MgATP (Km = 57 microM), and inhibition by increasing salt concentrations (I50 = 55 mM KCl). The protein kinase activity was not affected by either spermine, heparin, cAMP, cGMP or concentrations of CaCl2 less than 10 mM. The enzyme did not phosphorylate either phosphofructokinase or glycogen phosphorylase, two enzymes that are also phosphorylated during anoxia in whelks. The purified enzyme is different from the catalytic subunit of cAMP-dependent protein kinase as shown by the inability of cAMP to stimulate the protein kinase at all stages of the preparation; cAMP did not activate either crude enzyme, the 7% poly(ethylene glycol) supernatant, or any of the column eluant peak fractions when measured by changes in pyruvate kinase kinetic parameters.

Adenosine Triphosphate↗
PubMed1975

Membrane-localized pyruvate kinase of red blood cells in hemolytic anemia associated with pyruvate kinase deficiency.

Pyruvate kinase activity of red blood cell membranes, which is normally masked, has been determined after mechanical disruption of the membranes in normal individuals and in three homozygous patients with pyruvate kinase deficiency. Although patients 1 and 2, who were siblings, had relatively high enzyme activities in their hemolysates, they had the severest form of the disorder. The activities of their membrane fragments were decreased to seven per cent of fragments of normal membranes. Patient 3 had a mild form of hemolytic anemia despite a low enzyme activity of his hemolysates. The membrane fragments of this patient contained 28 per cent of the pyruvate kinase activity of normal fragments. The data suggest a relationship between the amount of membrane-localized pyruvate kinase and the severity of the clinical disorder. The reduced production of ATP by the enzyme portion localized within the membrane may cause an impairment of membrane functions in pyruvate kinase deficiency.

Aged↗
PubMed1996

Measurement of metabolic fluxes through pyruvate kinase, phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, and pyruvate carboxylate in hepatocytes of different acinar origin.

Isolated hepatocytes were prepared from the periportal and perivenous regions of the liver of 18-h-starved rats. These showed characteristics enzyme patterns and an enhanced rate of ureagenesis in the periportal cells; however, total cellular ATP content was unchanged in the two cell types. Measurements of pyruvate kinase flux showed no significant difference in the overall rate in the two cell types; however, the flux through phosphoenolpyruvate (PEP) carboxykinase was significantly higher in the periportal cells, such that the percentage of PEP being metabolized by pyruvate kinase was enhanced in the perivenous cells. The increase in partitioning of PEP through pyruvate kinase could account for only a small percentage of the difference in gluconeogenic flux in the two cell types, suggesting that the rate of provision of PEP was the principal limiting factor for glucose synthesis. The flux through pyruvate dehydrogenase showed no significant metabolic zonation, whereas pyruvate carboxylase flux was enhanced in the periportal zone. The partitioning of pyruvate between pyruvate carboxylase and pyruvate dehydrogenase was increase 2.8-fold in the periportal cells compared to that in the perivenous cells and it is suggested that this, together with possible alterations in phosphoenolpyruvate carboxykinase, is primarily responsible for the different gluconeogenic rates in the two zones of the liver.

Animals↗
PubMed1983

Purification and properties of pyruvate kinase from Streptococcus sanguis and activator specificity of pyruvate kinase from oral streptococci.

It was found that pyruvate kinases with two different regulatory characteristics were distributed among oral streptococci. The pyruvate kinases of Streptococcus mutans, Streptococcus salivarius, and Streptococcus bovis were activated by glucose 6-phosphate, whereas the enzymes of both Streptococcus sanguis and Streptococcus mitis were activated by fructose 1,6-bisphosphate. Pyruvate kinase (EC 2.7.1.40) from S. sanguis NCTC 10904 was purified, giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular weight of 250,000 to 260,000 and consisted of four identical subunits. Whereas the pyruvate kinase from S. mutans was completely dependent on glucose 6-phosphate (K. Abbe and T. Yamada, J. Bacteriol. 149:299-305, 1982), the enzyme from S. sanguis was activated by fructose 1,6-bisphosphate. In the presence of 0.5 mM fructose 1,6-bisphosphate, the saturation curves for the substrates, phosphoenolpyruvate and ADP, were hyperbolic, and the Km values were 0.13 and 0.30 mM, respectively. Without fructose 1,6-bisphosphate, however, saturation curves for both substrates were sigmoidal. GDP, IDP, and UDP could replace ADP. Like the enzyme from S. mutans, the enzyme from S. sanguis required a divalent cation, Mg2+ or Mn2+, and a monovalent cation, K+ or NH4+, for activity, and it was strongly inhibited by Pi. When the concentration of Pi was increased, the half-saturating concentration and Hill coefficient for fructose 1,6-bisphosphate increased. The remarkable fluctuation of intracellular levels of fructose 1,6-bisphosphate and phosphoenolpyruvate observed in the cells growing under glucose limitation and nitrogen limitation implies that the intracellular concentration of fructose 1,6-bisphosphate, in cooperation with that of Pi, may regulate pyruvate kinase activity in S. sanguis in vivo.

Cations↗
PubMed1978

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver.

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.

Erythrocytes↗
PubMed1977

Purification and characterization of pig kidney pyruvate kinase (type A).

Pyruvate kinase type A was purified from pig kidney with a yield of 9%. The final enzyme fraction had a specific activity of 500 units/mg of protein. The enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis in detergent and in ultracentrifugation experiments. The molecular weight of the enzyme was found to be 210,000 with the use of ultracentrifugation and 249,000 at gel chromatography. The sedimentation coefficient (S degrees 20, w) was calculated to be 9.8 S. For the reduced and alkylated pyruvate kinase, a molecular weight of 60,000 was found with the use of several methods. The Stokes radius for the enzyme was calculated to be 56 A. No NH2-terminal amino acid was detected in the enzyme, and the only findings in carbohydrate analyses of the kidney pyruvate kinase were trace amounts of glucose. The isoelectric point of the enzyme was estimated to be pH 5.6. Pig kidney pyruvate kinase type A was not phosphorylated on incubation with ATP and cyclic 3':5'-AMP-dependent protein kinase. The amino acid compositions of pig kidney and pig muscle pyruvate kinases were very similar and differed clearly from that of pig liver pyruvate kinase.

Amino Acid Sequence↗
PubMed1994

Metabolic depression in land snails: in vitro analysis of protein kinase involvement in pyruvate kinase control in isolated Otala lactea tissues.

Isolated tissues from the land snail Otala lactea were used to examine the relationship between protein kinase activity and phosphorylation-induced changes associated with metabolic depression. Hepatopancreas and foot muscle were removed from active and estivating land snails and incubated in vitro under aerobic and anoxic conditions. Pyruvate kinase (PK), cAMP-dependent protein kinase (PKA), and protein kinase second messenger compounds (cyclic AMP and inositol 1,4,5-triphosphate) were measured after incubating the tissues for 4 hours. Pyruvate kinase from the hepatopancreas of active snails was phosphorylated during anoxic incubations as indicated by changes in the I50 value for L-alanine. However, measurements of PKA activity and of cellular cAMP concentrations suggested that PKA activity was lower in these incubated tissues. When foot muscle was used as the tissue source, incubation under anoxic conditions produced no changes in PK activity even though PKA activity was drastically reduced. Analysis of changes in inositol 1,4,5-triphosphate concentrations after tissue incubation showed that they were not consistent with changes in PK activity in either organ. These results suggest that PKA and Ca2+/phospholipid-dependent protein kinase C do not phosphorylate PK during anoxia in land snails. The differences between values measured in incubated tissues and those measured in vivo suggest that isolated O. lactea tissues are not a good in vitro model system for studying metabolic changes associated with depressed metabolism.

Animals↗
PubMed2002

[From gene to disease; hereditary non-spherocytic hemolytic anemia caused by pyruvate kinase deficiency].

Pyruvate kinase (PK) deficiency is a common cause of hereditary non-spherocytic haemolytic anaemia. It is an autosomal recessive disorder caused by mutations in the gene coding for erythrocyte and liver-type pyruvate kinase (PKLR). So far, more than 130 mutations in this gene have been identified. Clinical symptoms, usually restricted to homozygous and compound-heterozygous individuals, are variable, ranging from neonatal jaundice requiring erythrocyte transfusions to a fully compensated haemolytic anaemia. The exact mechanism of erythrocyte destruction is unknown, however adenosine-triphosphate depletion and an increase in 2,3-disphosphoglycerate are thought to be important. The diagnosis of pyruvate kinase deficiency depends upon the demonstration of low PK enzyme activity. Due to the pitfalls in determining true PK activity, DNA testing is a valuable tool in the diagnosis of pyruvate kinase deficiency. By centralizing the molecular diagnostics of pyruvate kinase deficiency in Utrecht, more care can be provided for the diagnosis, treatment and support of patients.

Anemia, Hemolytic, Congenital Nonspherocytic↗
PubMed1988

Genetic and environmental components of serum creatine kinase (CK) and pyruvate kinase (PK) in normal twins: implication for genetic risks estimates in Duchenne muscular dystrophy carriers.

The serum activity of creatine kinase (CK) and pyruvate kinase (PK) was measured in 98 pairs of same-sex Brazilian twins. The purpose of this study was to estimate the genetic and environmental components of serum activity levels for both enzymes. Heritabilities were estimated separately by path analysis in each sex. The results showed that CK and PK activities are under genetic control in normal males and females. Environmental components were not statistically significant for CK or PK. The genetic component of both enzymes estimated in females has implications in the calculation of genetic risks for Duchenne muscular dystrophy carriers.

Creatine Kinase↗
PubMed1975

Electrophoretic, immunologic and kinetic characterization of erythrocyte pyruvate kinase in the Basenji dog with pyruvate kinase deficiency.

The electrophoretic mobility and the immunologic specificity of erythrocyte pyruvate kinase (PK) of the homozygous Basenji dog with PK deficiency were identical to those of normal M2-type PK isozyme seen in the white cell but not to those of the erythrocyte PK isozyme. Kinetic properties and stability were also consistent with the M2-type PK isozyme. Defective PK in the homozygous red cell was due to the absence of the erythrocyte PK isozyme and the compensatory presence of M2-type PK isozyme, as seen in the severe classical type PK deficiency in man.

Animals↗